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J. Biol. Chem., Vol. 281, Issue 14, 9607-9615, April 7, 2006

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Fulvestrant (ICI 182,780)-dependent Interacting Proteins Mediate Immobilization and Degradation of Estrogen Receptor-^*

Xinghua Long and Kenneth P. Nephew^¶1

From the Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405 and Department of Cellular and Integrative Physiology, Indiana University School of Medicine and ^¶Indiana University Cancer Center, Indianapolis, Indiana 46202

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor- (ER) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ER interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ER-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ER-interacting proteins, cytokeratins 8 and 18 (CK8·CK18). We determined, using ER-activating function-2 mutants, that helix 12 (H12) of ER, but not its F domain, is essential for fulvestrant-induced ER-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ER immobilization/degradation, transient transfection assays were performed using wild type ER,ER with a mutated H12, and ER with a deleted F domain. Of those, only the ER H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ER degradation in CK8·CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ER degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ER-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ER immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ER to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ER turnover.

Received for publication, October 4, 2005 , and in revised form, February 2, 2006.

^* This work was supported by The American Cancer Society Research and Alaska Run for Women Grant TBE-104125 and the United States Army Medical Research Acquisition Activity Awards DAMD 17-02-1-0418 and -0419. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 E. 3rd St., Bloomington, IN 47405-4401. Tel.: 812-855-9445; Fax: 812-855-4436; E-mail: knephew{at}indiana.edu.

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